ABSTRACTNucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)∼G(1)> G(2)). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G(2)∼G(1)> G(3), a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.

gkr1307-F1: (a) Chemical structures of AAF, FAAF, AF and FAF adducts; (b) sequences of fully paired 16-mer and 12-mer NarI duplexes used in the present study; major groove views of the central trimer segments of (c) the B/S and (d) B/S/W-conformer equilibrium of FAF and FAAF-modified duplexes. The modified dG and the complementary dC are shown in red and green sticks, respectively, and the aminofluorene moiety is highlighted with shiny gray CPK and the N-acetyl with pink CPK. In the B-type conformer, anti-[FAAF/FAF]dG maintains Watson–Crick hydrogen bonds, thereby placing the carcinogen moiety in the major groove. The carcinogens in the S- and W-conformers stack into the helix or wedged into the minor groove, respectively, with the modified dG in the syn conformation.

Mentions:
Arylamines are an important class of environmental pollutants that are implicated in the etiology of human cancers, especially of the bladder and liver (1). 2-Acetylaminofluorene was originally developed as an agricultural insecticide, but was later banned due to its strong tumorigenic activity in rat livers (8). It has been used extensively as a model for studying chemical carcinogenesis. In vivo, metabolic activation of AAF produces a highly electrophilic nitrenium ion, which subsequently interacts with DNA to produce two major C8-substituted dG adducts: AAF and AF (Figure 1a) (8,9). In vitro, N-acetylated AAF blocks the activity of high-fidelity polymerases and requires bypass polymerases for a translesion synthesis (TLS), whereas AF only slows down replication (10). In general, the bulky AAF exhibits greater susceptibility towards NER than AF (11), which is known to exist in a sequence-dependent equilibrium between anti B-conformer and syn S-conformer (Figure 1c) (10,12–14). We recently reported that AAF adducts also adopt a sequence-dependent S/B/W-conformational equilibrium (Figure 1d) (15).Figure 1.

gkr1307-F1: (a) Chemical structures of AAF, FAAF, AF and FAF adducts; (b) sequences of fully paired 16-mer and 12-mer NarI duplexes used in the present study; major groove views of the central trimer segments of (c) the B/S and (d) B/S/W-conformer equilibrium of FAF and FAAF-modified duplexes. The modified dG and the complementary dC are shown in red and green sticks, respectively, and the aminofluorene moiety is highlighted with shiny gray CPK and the N-acetyl with pink CPK. In the B-type conformer, anti-[FAAF/FAF]dG maintains Watson–Crick hydrogen bonds, thereby placing the carcinogen moiety in the major groove. The carcinogens in the S- and W-conformers stack into the helix or wedged into the minor groove, respectively, with the modified dG in the syn conformation.

Mentions:
Arylamines are an important class of environmental pollutants that are implicated in the etiology of human cancers, especially of the bladder and liver (1). 2-Acetylaminofluorene was originally developed as an agricultural insecticide, but was later banned due to its strong tumorigenic activity in rat livers (8). It has been used extensively as a model for studying chemical carcinogenesis. In vivo, metabolic activation of AAF produces a highly electrophilic nitrenium ion, which subsequently interacts with DNA to produce two major C8-substituted dG adducts: AAF and AF (Figure 1a) (8,9). In vitro, N-acetylated AAF blocks the activity of high-fidelity polymerases and requires bypass polymerases for a translesion synthesis (TLS), whereas AF only slows down replication (10). In general, the bulky AAF exhibits greater susceptibility towards NER than AF (11), which is known to exist in a sequence-dependent equilibrium between anti B-conformer and syn S-conformer (Figure 1c) (10,12–14). We recently reported that AAF adducts also adopt a sequence-dependent S/B/W-conformational equilibrium (Figure 1d) (15).Figure 1.

ABSTRACTNucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)∼G(1)> G(2)). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G(2)∼G(1)> G(3), a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.